Fig. 2. Structure-based rational design of covalent inhibitors of PPARγ phosphorylation. (a) The proposed specific binding site in the SR1664–PPARγ LBD X-ray co-crystal structures (green). (b) The specific binding site on the surface of the PPARγ LBD complexed with GW9662 (black, PDB: 3B0R). (c) Structure-based design of the covalent inhibitors SB1405 and SB1406, which were expected to bind at the specific site. (d, e) In vitro Cdk5 assay of PPARγ or the Rb peptide on treatment with rosiglitazone, SR1664, or the covalent inhibitors. NT, no treatment; pPPARγ, phosphorylated PPARγ; pRb peptide, phosphorylated Rb peptide. (f, g) Binding modes of SB1405 (pink) and SB1406 (brown) confirmed by X-ray crystallography. (h) Chemical structures of N-(2-substituted phenyl)-2-chloro-5-nitrobenzamides. (i, j) In vitro Cdk5 assay of PPARγ or the Rb peptide on treatment with rosiglitazone or N-(2-substituted phenyl)-2-chloro-5-nitrobenzamides.